Lay Summary

There is an urgent need for better therapies to treat patients with the most aggressive type of childhood cancer, Diffuse Intrinsic Pontine Glioma (DIPG). DIPG is an incurable brain cancer with 5-year survival of <1%. We have formed an international collaboration to find genetic causes of DIPG, and have made the important discovery that a subset of DIPG patients have hereditary mutations in genes that are known to predispose to breast and ovarian cancers, such as BRCA1 and BRCA2. These mutations prevent the cells from effectively repairing damaged DNA, which can result in cancer progression. Fortunately, BRCA mutations can be targeted with existing drugs called poly(ADP-ribose) polymerase (PARP) inhibitors, and these drugs are already being used clinically for the treatment of adult cancers. Thus, PARP inhibitors represent a new way for treating DIPG patients who carry similar BRCA mutations. Here, we will study the behaviour of BRCA-mutant DIPG cells and determine if PARP inhibitors can halt the growth of these cells. As PARP inhibitors have been suggested to sensitize cancer cells to DNA-damaging radiation, we will also test PARP inhibitors with standard-of-care radiotherapy to obtain maximum efficacy. This work has the potential to lead to the first effective treatment option for children with DIPG.

Executive Summary

Background:

DIPG is the most aggressive of all childhood cancers.  It is a type of brain tumor that peaks in incidence at 5-7 years of age and is the most common form of malignant glioma to affect children. There are no effective treatments and current therapeutic strategies are palliative only, with 99% of patients dying within 5 years. Due to its location within the brainstem, the tumor cannot be removed surgically, it does not respond to chemotherapy, and radiotherapy only slows cancer growth temporarily. Novel and innovative treatment approaches are therefore urgently needed to counter this tumor.

The rate of germline pathogenic mutations across the spectrum of pediatric cancers is higher than previously appreciated. We have shown that 16% of children with high-risk cancers enrolled on the Australian Zero Childhood Cancer National Precision Medicine Trial (ZERO) were found to have a mutation in a known cancer predisposition gene [1]. Our research team initiated an international collaboration spanning USA, Canada, UK, Germany, and Australia and assembled a cohort of >300 DIPG patients with both germline and somatic sequencing data and discovered that a significant proportion (~10%) of these patients harbored mutations in the Homologous Recombination (HR) DNA repair pathway, such as in the BRCA1 and BRCA2 tumor suppressor genes. The HR repair pathway involves a complex cascade of events, ending in a recombinase reaction using a homologous DNA template for high-fidelity repair of the damaged DNA [13]. Germline mutations in BRCA1 and BRCA2 are well known for increasing susceptibility to breast and ovarian cancers, and other malignancies such as pancreatic, prostate, colorectal and stomach cancer [2]. In these cancer types, BRCA1/2 mutations result in defective HR-mediated DNA repair, increased genomic instability and oncogenic transformation. However, the contribution of BRCA deficiency to DIPG pathogenicity is not yet understood. We expect that BRCA1/2 mutations will increase DIPG tumorigenicity.  

BRCA-mutant cancer cells are sensitive to PARP inhibitor drugs, leading to the FDA approval of olaparib, rucaparib, niraparib, and talazoparib for patients with HR-deficient cancers [3]. Whether such a relationship exists for BRCA-mutant DIPG has not been tested. Of the four FDA-approved PARP inhibitors, niraparib and olaparib have demonstrated blood-brain-barrier permeability [4, 5]. Importantly, PARP inhibitors can radiosensitize breast cancer cells [6]. This is relevant given that radiotherapy is the only current standard-of-care treatment for DIPG. We have preliminary data to suggest that BRCA loss leads to increased sensitivity to both niraparib and irradiation. Whether this exciting result will be recapitulated across more models of DIPG, and in vivo, remains to be discovered and forms the basis for this proposal. Strikingly, one DIPG patient enrolled on ZERO, who had a germline BRCA2 mutation, was treated with the PARP inhibitor olaparib in combination with a checkpoint inhibitor after disease progression, leading to a sustained complete response – an extraordinary outcome in this highly aggressive malignancy.

Hypothesis:

We hypothesize that BRCA1/2-mutant DIPG represents a new subtype of patients, driven by HR deficiency and increased genomic instability, and that these patients will respond favorably to PARP inhibition.

Design and methodology:

Aim 1: Develop and characterize patient-derived DIPG cell line models with BRCA1/2 depletion:

First, we will develop the first models of DIPG with BRCA1/2 loss-of-function, using lentiviral transduction of inducible shRNA constructs targeting BRCA1 and BRCA2. Two individual shRNA sequences per gene will be used, and a scrambled non-targeting shRNA will serve as a negative control to account for off-target RNAi effects. Three primary DIPG cultures will be transduced, which have differing H3K27M and p53 mutational status to account for clinical heterogeneity and to investigate the relationship between BRCA loss and other aberrations known to be involved in genomic instability and the DNA damage response. These cells will be characterized using in vitro assays to assess their HR repair capability (DNA damage foci and the alkaline comet assay), as well as interrogation of any arising mutational signatures by whole genome sequencing (WGS).

Aim 2: Determine the impact of BRCA depletion on DIPG tumorigenesis

Next, we will determine the contribution of BRCA1/2 loss to DIPG tumorigenicity. To that end we will measure i) the proliferation and clonogenic potential of the BRCA1/2 knockdown cells in vitro, and ii) the ability of these cells to form tumors in vivo when injected orthotopically into the brainstem of mice. In addition to the established human DIPG cultures, we will also knockdown BRCA1/2 in an in vivo model of tumor progression using in utero electroporation of murine embryonic neural precursors with vectors containing shRNA for BRCA1/2, along with other common DIPG drivers such as p53 loss, H3K27M, and PDGFRA overexpression. These studies will reveal whether BRCA mutations contribute to the oncogenic transformation of neural precursors into DIPG.

Aim 3: Determine the therapeutic efficacy of PARP inhibitors and irradiation in cell and animal models of DIPG with BRCA1/2 depletion

Finally, we will assess the sensitivity of the BRCA1/2 knockdown models to PARP inhibitors and irradiation. Human DIPG cells with BRCA1/2 depletion will be treated with PARPi and irradiation as single agents and in combination. The effect on cell viability and colony formation will be assayed. Finally, these cells will be orthotopically injected into mice and treated with PARPi and irradiation to determine the therapeutic efficacy of these treatments in vivo.

Expertise and feasibility:

Our skilled team of biologists, bioinformaticians and clinicians have all the necessary expertise that will ensure the success of the proposed project and ultimately the implementation of the discoveries into the clinic. Lead-Investigator Dr Holly Holliday is a postdoctoral researcher with relevant expertise in both breast cancer and DIPG research and has generated the background data in this proposal. Dr Holliday will be responsible for generating the remaining BRCA1/2 knockdown models and for performing the in vitro experiments. The Children’s Cancer Institute Bioinformatics team, led by Chelsea Mayoh, has extensive expertise in WGS analysis required for characterization of mutational signatures in these models. Dr Maria Tsoli is a senior scientist with specific expertise in DIPG cell cultures and xenograft models, drug combinations and mechanistic studies and will be responsible for supervising the in vivo experiments. We have established collaborations with Prof Nada Jabado (McGill University, Montreal, Canada) who will provide expert guidance with the in utero electroporation study [7]. Dr Marion Mateos is a clinician scientist who has curated the germline patient data from the ZERO cohort and from international collaborators: Dr Chris Jones (ICR, UK), Dr David Jones & Prof Stefan Pfister (DKFZ, Heidelberg, Germany), Prof Nada Jabado (McGill University, Montreal, Canada) and Dr Adam Resnick (CHOP, Philadelphia, USA). Dr Mateos’ experience in pre-clinical development of new therapeutics will guide the project’s development and aid in clinical translation. Prof Ziegler has pre-clinical expertise in pediatric malignant brain tumors and his clinical focus on early phase clinical trials will facilitate translation of positive results to the bedside. The support of Prof Ziegler and Prof Haber at Children’s Cancer Institute provides an invaluable environment to ensure the success of this novel research program, with the ultimate goal of improving outcomes for children diagnosed with DIPG.

Outcomes and clinical significance:

In Aim 1, we will establish the first models of BRCA-mutant DIPG, characterise their capacity for HR-mediated repair of DNA lesions, and assess their mutational burden by whole genome sequencing. We expect that, like in other malignancies, BRCA loss-of-function will impair HR repair and increase mutational burden, and that this will be further exacerbated in cells containing H3K27M and p53 mutations. In Aim 2 we will determine if loss of BRCA function is sufficient to increase the tumorigenic potential of DIPG cells, and to transform early neural progenitors. We anticipate that HR deficiency will be pivotal in promoting DIPG pathogenicity. In Aim 3, the efficacy of PARP inhibitors and irradiation at inhibiting proliferation, colony formation and tumor growth in primary cultures and animal models will be established. We expect that these pre-clinical experiments will demonstrate that the combination of PARP inhibition with standard-of-care radiotherapy is effective for DIPG patients with HR deficiency.

DIPG is currently an incurable brain cancer. We have preliminary genomic, in vitro and clinical data that suggest that there is a specific subset of patients who may respond to PARP inhibitors. By the end of this project, we expect to demonstrate that BRCA mutations are a key contributor to DIPG tumorigenicity and that targeting these mutations with PARP inhibitors and irradiation will demonstrate therapeutic efficacy in vitro and in mouse models. Our discoveries have the potential to be rapidly translated into clinical trials given that brain-penetrant PARP inhibitors are already in clinical use and readily accessible, and that radiotherapy is the current standard of care for DIPG. If successful, this study could provide the essential pre-clinical data that would enable the repurposing of PARP inhibitors for this subset of DIPG, and by extension, other pediatric cancers with HR deficiencies.  

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest